Inhibitors and methyl donor substrates for BHMT were used to study the role of this enzyme in regulating tHcy. Fasted mice were given an intraperitoneal injection of S-(delta-carboxybutyl)-DL-homocysteine (CBHcy; 1 mg), a specific and potent inhibitor of BHMT, and their tHcy and hepatic BHMT protein and activity levels were monitored over a 24 h period. Compared to saline-injected control mice, at 2 h post-injection the CBHcy-treated mice had 87% lower hepatic BHMT activity and a 3.7-fold increase (11.1 vs. 3.0 uM) in tHcy; effects that lasted nearly 8 h but returned to normal by 24 h. The level of hepatic BHMT protein remained constant over the 24 h period, independent of treatment. Two h after injecting the sulfoxide derivative of CBHcy (10 mg) into fasted mice there was a modest reduction of hepatic BHMT activity and a 1.9-fold increase in tHcy. When given an injection of Met (3 mg) or Met plus CBHcy (1 mg), post-Met load tHcy levels were 3.2-fold higher (128 vs. 40 uM) at 2 h post-injection in the mice given CBHcy. CBHcy does not inhibit folate-dependent methionine synthase, or pyridoxal phosphate-dependent cystathionine synthase. Like betaine, dimethylsulfoniopropionate was an effective tHcy-lowering agent when given with a Met load. These studies are the first to show that transient inhibition of BHMT in vivo causes transient hyperhomocysteinemia, and that dimethylsulfoniopropionate can reduce the post-Met load rise in tHcy. Supported by NIDDK (DK52501) and IL ARS (50–352) to TAG, and grants from the Czech Academy of Sciences (A4055302) and Research Project (Z40550506) to JJ.
Betaine-homocysteine S-methyltransferase (BHMT) transfers a methyl group from betaine to Hcy to form DMG (dimethylglycine) and Met. The reaction is ordered Bi Bi; Hcy is the first substrate to bind and Met is the last product off. Using intrinsic tryptophan fluorescence [Castro, Gratson, Evans, Jiracek, Collinsova, Ludwig and Garrow (2004) Biochemistry 43, 5341–5351], it was shown that BHMT exists in three steady-state conformations: enzyme alone, enzyme plus occupancy at the first substrate-binding site (Hcy or Met), or enzyme plus occupancy at both substrate-binding sites (Hcy plus betaine, or Hcy plus DMG). Betaine or DMG alone do not bind to the enzyme, indicating that the conformational change associated with Hcy binding creates the betaine-binding site. CBHcy [S-(δ-carboxybutyl)-D,L-homocysteine] is a bisubstrate analogue that causes BHMT to adopt the same conformation as the ternary complexes. We report that BHMT is susceptible to conformation-dependent oxidative inactivation. Two oxidants, MMTS (methyl methanethiosulphonate) and hydrogen peroxide (H2O2), cause a loss of the enzyme's catalytic Zn (Zn2+ ion) and a correlative loss of activity. Addition of 2-mercaptoethanol and exogenous Zn after MMTS treatment restores activity, but oxidation due to H2O2 is irreversible. CD and glutaraldehyde cross-linking indicate that H2O2 treatment causes small perturbations in secondary structure but no change in quaternary structure. Oxidation is attenuated when both binding sites are occupied by CBHcy, but Met alone has no effect. Partial digestion of ligand-free BHMT with trypsin produces two large peptides, excising a seven-residue peptide within loop L2. CBHcy but not Met binding slows down proteolysis by trypsin. These findings suggest that L2 is involved in the conformational change associated with occupancy at the betaine-binding site and that this conformational change and/or occupancy at both ligand-binding sites protect the enzyme from oxidative inactivation.
Betaine homocysteine methyltransferase (BHMT) and BHMT‐2 methylate homocysteine using betaine or S‐methylmethionine, respectively. These enzyme activities are found only in the liver of adult rodents, but in the liver and kidney of adult humans and pigs. Using a pig model, we seek to understand the developmental expression pattern of these enzymes in different porcine organs. Immunohistochemical staining revealed the presence of BHMT in adult liver and kidney cortex, as reported earlier, and also in fetal lungs (aged days 30, 60, 84, 90, 105). Full length cDNA of BHMT and BHMT‐2 were cloned and sequenced, and the 5' and 3' UTRs were amplified using RLM‐RACE. BHMT has a longer 5' and 3' UTR, consisting of 77 and 1142 nucleotides, whereas BHMT‐2 consisted of 17 and 893 nucleotides respectively. The predicted primary sequences of BHMT and BHMT‐2 contain 407 and 363 amino acids, respectively, and share 78% amino acid identity. Relative to BHMT‐2, BHMT has two additional regions of amino acid sequence; a 9 amino acid sequence (86‐94) in the N‐terminal region, and 34 amino acid sequence (373‐407) at the carboxy terminus. Preliminary sequencing results identified two isoforms of liver BHMT gene resulting from alternative splicing of 5' UTR. BHMT enzymes are organ/developmental stage‐dependent and further sequence analysis might reveal new splice variants that result in different protein functions. Grant Funding Source USDA AG 2008‐34480‐19328 & USDA‐ARS 538 AG58‐5438‐7‐317l
